The present invention relates to the purification of a protein produced by recombinant cells. Specifically, it provides a process for the efficient purification of hepatitis surface antigen (HBsAg) from cultures of recombinant cells producing this protein, particularly mammalian recombinant cells.
Viral hepatitis B is transmitted by carriers of hepatitis B through blood and body secretions. HBsAg has been purified from blood serum and made into a vaccine. Purcell, R. H. and Gerin, J. L. in Viral Hepatitis, The Franklin Institute Press (1978) p. 491 and Hilleman, M. R. et al ibid. p. 525. This publication describes a procedure for isolation of HBsAg in which the plasma from hepatitis B carriers is defribinated, and the HBsAg in the serum is (1) concentrated by ammonium sulfate precipitation, (2) subjected to isopycnic banding in a centrifuge using sodium bromide as the gradient medium, (3) subjected to rate-zonal sedimentation using a sucrose gradient, (4) inactivated with a variety of steps, (5) subjected to gel filtration, and (6) produced into a vaccine. Use of serum as a source for HBsAg is dangerous because of the presence of hepatitis B virus as well as the presence of as yet undefined adventitious agents. In addition, the techniques employed for purification of the HBsAg are time consuming and complicated. Formulation of hepatitis surface antigen for vaccine purposes has been performed by compounding with aluminum adjuvants such as aluminum hydroxide (op cit. Viral Hepatitis) and aluminum phosphate as described by Reerink-Brongers. E. E., et al, Developments in Biological Standardization, S. Karger (Basel, Switzerland) (1983) 54, 197.
Purification of a protein such as HGsAg produced in a recombinant cell may require different techniques from those which are used to purify HBsAg from "natural" sources such as blood serum due to the significantly different compositions of the two sources. Thus, one cannot predict that a technique used for purification of HBsAg from natural sources would be useful for purification of HBsAg from recombinant cell sources. Predictability is diminished even further where the final product must be exceptionally pure, e.g. for use as a human vaccine.
The present invention is directed to a process based upon chromatography which is successful in isolating HBsAg from recombinant cell cultures. A unqiue sequence of fractionation techniques is performed to produce HBsAg of high purity for use in human vaccine. This product is free of active adventitious agents.